PROJECT SUMMARY/ABSTRACT Therapeutic modulation of immune checkpoints has emerged as a transformative new paradigm in the treatment of cancer. This development has triggered immense interest in targeting new co-regulatory pathways to amplify the early success of immunotherapy. A major challenge in this endeavor is the widespread pleiotropy characteristic to immune signaling pathways. Due to the promiscuity of their ligand:receptor interactions, co-regulatory proteins can produce multiple, often contradictory activities. This poses difficulty in developing agents that can isolate specific pathway functions and obscures a precise understanding of the physiology of individual receptor:ligand pairs. The LIGHT/HVEM/LT?R signaling network is a key exemplar of this challenge. LIGHT is an immune co-stimulator that plays a crucial role in the activation and expansion of T cells. It signals through both the Herpes virus entry mediator (HVEM) and the lymphotoxin beta receptor (LT?R), and it is antagonized by decoy receptor 3 (DcR3). However, each of these receptors binds additional ligands besides LIGHT, resulting in a complicated and interconnected signaling network. Here, we propose to leverage our expertise in structure-guided protein engineering to develop and characterize LIGHT variants that can uncouple the disparate activities of the LIGHT/HVEM/LT?R pathway. We will knock-in these ?biased? alleles into transgenic mice to study individual pathway interactions in vivo and in parallel, we will assess the therapeutic potential of biasing LIGHT activity in models of immunotherapy. In sum, this proposal will provide insight into a pivotal immunoregulatory pathway, candidate molecules for therapeutic development, and new approaches for targeting pleiotropic checkpoint receptors.